Unphosphorylated STAT recruits HP1 for gene repression Abstract Aberrant activation of JAK/STAT signaling is associated with many types of human cancers. The effects of JAK/STAT signaling have been attributed largely to direct transcriptional regulation by activated or phosphorylated STAT (pSTAT) of specific target genes that promote tumor cell proliferation and/or survival. Inhibitory effects of unphosphorylated STAT (uSTAT) on cell proliferation have been previously reported and were assumed to be due to its interference with the transcriptional activity of pSTAT. Moreover, uSTAT has been considered to be the latent form of STAT that resides in the cytoplasm. Many groups have reported, however, that uSTAT is also found in the nucleus. Our research in both Drosophila and human cells has demonstrated a non-canonical mode of JAK/STAT signaling, in which a fraction of uSTAT resides in the nucleus in association with Heterochromatin Protein 1 (HP1); STAT activation (by phosphorylation) causes its dispersal from heterochromatin, which leads to HP1 delocalization and heterochromatin loss. We have further shown that human uSTAT5A binds to HP1?, stabilizes heterochromatin, and inhibits cancer growth in a mouse xenograft model. We have found that expressing uSTAT5A has similar effects to overexpressing HP1? on global gene expression in colon cancer cells, and that the majority of the genes commonly repressed by U-STAT5A and HP1? are overexpressed or important for colorectal cancer development, whereas knocking down endogenous STAT5A or HP1? derepresses these genes. These results suggest that uSTAT5A may function as a tumor suppressor, via recruiting HP1, by repressing genes important for cancer development or growth. We hypothesize that nuclear uSTAT is capable of recruiting HP1 to chromatin for gene repression. In this proposal, we will test this hypothesis by investigating the ability of both Drosophila uSTAT and human uSTAT5A in recruiting HP1 proteins to chromatin and in transcriptional repression. Results from these studies should advance our understanding of the functions of JAK/STAT signaling components with implications in cancer treatment.